The formation and dissolution of blood clots (thrombi) are the basic processes involved in the physiological response to blood vessel injury. Blood vessel injury triggers a complex and highly regulated series of events that culminates in the cleavage of fibrinogen to fibrin monomers which spontaneously polymerize to form the matrix of the blood clot (Colman et al., "Overview of Hemostasis", in: Hemostasis and Thrombosis; Basic Principles and Clinical Practice, 2nd edition, J. B. Lipptncott Co., Philadelphia, 1987, pp. 3-17). Fibrinogen is cleaved by thrombin, the activated form of the zymogen prothrombin. Fibrinogen is a six chain molecule consisting of two A.alpha. chains, two B.beta. chains and two .gamma. chains; cleavage by thrombin results in the release of fibrinopeptides A and B (FPA and FPB) from the amino-termini of the A.alpha. and B.beta. chains. Thrombin also activates Factor XIII.sub.a which cross-links the fibrin clot and increases its resistance to fibrinolysis. Circulating thrombin is inactivated by antithrombin III, thus, preventing systemic coagulation of the blood.
A small amount of plasma plasminogen binds to or is incorporated in the fibrin clot. Plasminogen is also a zymogen and its activated form, plasmin, is the main agent of fibrinolysis. Plasminogen is activated by plasminogen activators which are secreted from endothelial cells or other organs. Circulating plasmin does not prevent clot formation due to inhibition by .alpha..sub.2 -antiplasmin; however, plasmin formed on the surface of the clot appears to be protected from inhibition and degrades fibrin. The inhibitory activities of antithrombin III and .alpha..sub.2 -antiplasmin localize the coagulative and fibrinolytic processes to the wound site. Dissolution of the clot is part of the recovery phase and prepares for endothelial cell regrowth and vessel recanalization.
The processes of clot formation and dissolution are carried out and regulated by a complex hemostatic system which includes platelets, endothelial cells, adhesive proteins, and numerous zymogen-activators and protein inhibitors. The concerted action of this system determines a delicate balance between the antagonistic processes of clot formation and dissolution. Thus, it is not surprising that many pathological disorders arise from dysfunction in this system.
The pathological condition characterized by dysfunctional formation of blood clots is referred to as thrombosis. Thrombosis is a pathogenic component of many cardiovascular disorders, including ischaemic heart disease (myocardial infarction and sudden coronary death), stroke, and peripheral vascular disorders, such as deep-vein thrombosis and thrombophlebitis (Marder and Sherry, N.E.J. of Med. 318(24):1585-1595 (1988); Cook and Ubben, Trends in Pharmacological Science 11:444-451 (1990)). Elevated plasma fibrinogen has been found to have a stronger association with ischaemic heart disease than blood cholesterol levels (Meade et al., The Lancet ii:533-537 (1986)) and also appears to be a significant diagnostic factor for the severity of atherosclerosis. In addition, high fibrinogen appears to be related to cancer, inflammatory disease, and the patency of transplant grafts. The pathological mechanism of thrombosis is not well understood.
Clinical treatment of thrombosis has consisted mainly of administering thrombolytic agents intravenously or locally by catheter (see Marder and Sherry, N.E.J. of Med. 318(24):1512-1520 and 318(24):1585-1595 (1988) for review). The thrombolytic agents currently used: streptokinase, acylated plasminogen-streptokinase activator complex (APSAC), tissue type plasminogen activator (t-PA), and urokinase plasminogen activator (u-PA), are all plasminogen activators. Urokinase plasminogen activator, a two chain protein, is usually administered as the recombinant pro-urokinase or single chain form (scu-PA), which forms a two chain urokinase (tcu-PA) on the clot. t-PA and scu-PA have been shown, in vitro and in animal models, to be fibrin-specific, i.e., they selectively activate fibrin-bound plasminogen while leaving systemic plasminogen mainly unaffected. Thrombolytic therapy with t-PA or scu-PA, however, suffers significant shortcomings. The therapeutic dose of these plasminogen activators has been found to be high, leading to a loss of fibrin-specificity and some systemic fibrinolytic activity, as evidenced by a drop in plasma fibrinogen levels upon dosage. As a result, thrombolytic therapy with t-PA and scu-PA is often complicated by hemorrhaging.
Some attempts have been made to improve thrombolytic therapy by increasing the fibrin-specificity and/or thrombolytic activity of t-PA and scu-PA. One suggested improvement is combining t-PA and scu-PA. This is based on the possible synergism between the thrombolytic activities of t-PA and scu-PA. This synergism has been explained by the different mechanisms of action of the two plasminogen activators (Pannell et al., J. Clin. Invest, 81:853-859 (1988)). t-PA binds strongly to fibrin, thus, forming a complex with the plasminogen also bound to the fibrin clot. scu-PA, on the other hand, does not bind significantly to fibrin clots in plasma. Its selectivity for fibrin-bound plasminogen apparently results from a conformational change in plasminogen upon binding to fibrin which renders it more sensitive to activation by scu-PA. t-PA and scu-PA seem to activate different species of plasminogens bound to the fibrin clot. t-PA acts on plasminogens bound to internal lysine residues of fibrin, while scu-PA acts on plasminogens bound to terminal lysine residues. Thus, the t-PA and scu-PA activities complement each other and together, have increased thrombolytic activity without decreased fibrin-specificity.
Another suggested improvement is enhancement of scu-PA therapy with plasminogen (U.S. Pat. No. 4,996,050, by Tsukada et al., Feb. 26, 1991). Plasminogen has been found to increase the fibrinolytic activity of scu-PA without causing increased systemic fibrinolysis (i.e., a drastic drop in plasma fibrinogen levels). Enhancement of scu-PA activity permits lowering the dose of scu-PA and may result in less side effects.
A further suggested development of plasminogen enhancement is supplementation with lys-plasminogen in place of glu-plasminogen (EP 307,847, by Kakkar et al., Mar. 22, 1989; Watahiki et al., Thrombosis and Haemostasis 61:502-506 (1989)). Lys-plasminogen is an 8 kDa degraded form of glu-plasminogen (92 kDa), with the NH.sub.2 -terminus at Lys.sub.77. It does not normally occur in plasma and is generated in small amounts during thrombolytic therapy. Lys-plasminogen has been found to be more sensitive to activation by tcu-PA and scu-PA and to have a higher affinity for fibrin clots than native glu-plasminogen. Supplementation with lys-plasminogen was found to result in greater enhancement of scu-PA activity than supplementation with glu-plasminogen. Glu-plasminogen was slightly more effective than lys-plasminogen at enhancing fibrinolysis by scu-PA/t-PA mixtures. Addition of low concentrations of tcu-PA has also been shown to have a synergistic effect on scu-PA activity. Glu- and lys-plasminogen were found to have equivalent enhancement of scu-PA/tcu-PA mixtures. Enhancement by lys-plasminogen was not accompanied by a significantly greater decrease in fibrinogen than observed with glu-plasminogen.